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Updated: Dec 16, 2025
Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells
Published on: April 27, 2018
Reversible inhibition of hydrogen peroxide elimination by calcium in brain mitochondria
Laszlo Tretter1, Emanuela Biagioni Angeli, Mohammad Reza Ardestani
1Department of Medical Biochemistry, Semmelweis University, and Laboratory of Neurobiochemistry and Molecular Physiology, Hungarian Academy of Sciences, Budapest, Hungary.
Abstract:
In the present work, the Ca(2+) dependence of mitochondrial H(2) O(2) elimination was investigated. Mitochondria isolated from guinea pig brain were energized by glutamate and malate and incubated with micromolar concentrations of Ca(2+) in the presence of ADP, preventing permeability transition pore formation. After the completion of Ca(2+) uptake, mitochondria were challenged with H(2) O(2) (5 μM), then at various time points residual H(2) O(2) was determined using the Amplex red method and compared with that in mitochondria incubated with H(2) O(2) without Ca(2+) addition. Dose-dependent inhibition of H(2) O(2) elimination by Ca(2+) was detected, which was prevented by the Ca(2+) -uptake inhibitor Ru 360. Stimulation of Ca(2+) release from Ca(2+) -loaded mitochondria by a combined addition of Ru 360 and Na(+) decreased the Ca(2+) -evoked inhibition of H(2) O(2) removal. After Ca(2+) uptake (50 μM), mitochondrial aconitase activity was found to be decreased, which was partially attributable to the impaired elimination of endogenously produced reactive oxygen species. We found that the effects of Ca(2+) and H(2) O(2) on the activity of aconitase were additive. These results confirm that Ca(2+) inhibits elimination of H(2) O(2) in mitochondria and demonstrate that this effect is concentration dependent and reversible. The phenomenon described here can play a role in the modulation of ROS handling under conditions involving excessive cellular Ca(2+) load.
Insights
Calcium ions (Ca2+) inhibit mitochondrial hydrogen peroxide (H2O2) removal in a dose-dependent manner. This reversible effect impacts reactive oxygen species (ROS) handling, particularly during high cellular Ca2+ conditions.
Area of Science:
- Mitochondrial Physiology
- Cellular Redox Biology
- Biochemistry
Background:
- Mitochondria are key regulators of cellular calcium (Ca2+) homeostasis.
- Mitochondrial reactive oxygen species (ROS) production and elimination are critical for cell function.
- Dysregulation of Ca2+ and ROS metabolism is implicated in various pathologies.
Purpose of the Study:
- To investigate the impact of Ca2+ on mitochondrial hydrogen peroxide (H2O2) elimination.
- To determine the concentration-dependence and reversibility of Ca2+-mediated inhibition of H2O2 removal.
- To explore the consequences of impaired H2O2 elimination on mitochondrial enzyme activity.
Main Methods:
- Isolation of guinea pig brain mitochondria.
- Energization of mitochondria using glutamate and malate.
- Incubation with varying Ca2+ concentrations and ADP to prevent pore formation.
- Measurement of H2O2 levels using the Amplex red assay.
- Assessment of mitochondrial aconitase activity.
- Utilizing Ca2+ uptake inhibitor (Ru 360) and Na+ for Ca2+ release studies.
Main Results:
- Ca2+ significantly inhibited mitochondrial H2O2 elimination in a dose-dependent manner.
- The inhibitory effect of Ca2+ was reversible upon Ca2+ removal.
- Ca2+ uptake led to decreased mitochondrial aconitase activity, partly due to impaired ROS elimination.
- The effects of Ca2+ and H2O2 on aconitase activity were additive.
Conclusions:
- Ca2+ reversibly inhibits mitochondrial H2O2 elimination.
- This Ca2+-dependent modulation of ROS handling can be significant under conditions of excessive cellular Ca2+.
- The findings provide insights into mitochondrial redox regulation and its potential role in disease states involving Ca2+ overload.
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